Characterization of the purified cGMP-stimulated cyclic nucleotide phosphodiesterase has been carried out. Catalytic activity of the enzyme required divalent cations. The relative effectiveness of Mg-2+, Mn-2+ and Co-2+ depended on the concentrations of cyclic nucleotide present as substrate or effector. Substitution of Mn-2+ for Mg-++ increased apparent Km (15 to 45 MuM) and reduced Vmax (200 to 160 Mumol/min/mg) for cGMP but had little effect on Km or Vmax for cAMP (Km: 33 vs. 47 MuM, Vmax: 170 vs. 170 Mumol/min/mg). Co-2+ increased Km and reduced Vmax for both cAMP and cGMP (70 MuM and 90 Mumol/min/mg for cAMP, 35 MuM and 130 Mumol/min/mg for cGMP). cGMP stimulated cAMP hydrolysis by about 32-fold in the presence of Mug-2+ but only 9-fold with Mn-2+ or 6-fold with Co-2+. Neither Co-2+ nor Mn-++ increased the apparent Ka for stimulation of cAMP hydrolysis. In the presence of optimal Mg-2+, phosphate increased enzyme activity and altered the homotropic effects of cAMP and cGMP. In the presence of phosphate, kinetics of cAMP and cGMP hydrolysis were quite similar and not suggestive of positive cooperativity. The apparent Km and Vmax for cAMP and for cGMP were markedly increased but identical (Km: 700 MuM, Vmax: 1.2 mmol/min/mg, Hill coefficient: 0.89). Taken together, these data suggest that not only cyclic nucleotides but also divalent cations and phosphate affect cooperative interactions of the cGMP-stimulated cyclic nucleotide phosphodiesterase. Mn-2+ seems to selectively affect cGMP-induced responses. On the other hand, phosphate, which presumably caused changes in enzyme configuration with produced enhanced catalytic activity toward cyclic nucleotides, also reduced homotropic responses to cAMP and cGMP and an alteration in those determinants responsible for discrimination between cAMP and cGMP.